Preserved in a horse’s hair, on a molecular level, is a chemistry trail that can tell a story of mistreatment or fair play.

As of July 1, the U.S. Equestrian Federation board of directors has implemented a rule adding hair sample testing to its anti-doping toolkit. This technology can detect substances that evade blood and urine tests—including barbiturates, which are typically seen only in euthanasia cases. In the rule change, the USEF board wrote it had expanded its testing “in response to member feedback that some trainers are misusing prohibited substances to gain a competitive edge at the expense of the horse’s health and well-being.”  

Petra Hartmann is the director of drug testing services at Industrial Laboratories Co. Inc. in Arvada, Colorado, which processes drug testing for regulatory bodies for performance animals, including testing for horse racing, greyhound racing and USEF competitions. She has a background in forensic toxicology and forensic drug testing, and she is also a fellow-level member of the Association of Official Racing Chemists, an international organization of laboratory professionals that work in anti-doping science. The lab will apply the same testing protocols it has long used for race horses to the influx of samples from USEF. 

“I would say currently, in race horses, we are testing well over 20,000 samples a year,” Hartmann said. “It’s a lot of hair. As a matter of fact, we have an area of our laboratory that they call ‘the salon’ because it’s solely dedicated to hair testing at this point.”

Hartmann spoke with the Chronicle about the capabilities and limitations of a science that may be new to many USEF members and horse owners but is well established in the broader performance animal industry.

What is the timeline for processing hair samples? How quickly are you getting results back to the regulators?

The standard turnaround time, generally speaking for race horses, is seven days plus seven business days: so seven days for screening the sample, and then an additional seven days to conduct any sort of confirmatory testing following suspect findings in the initial test. 

The hair test, compared to a lot of the other analytical testing that we do on blood and urine samples, is still a lot more manual and labor intensive. So while we have a pretty large staff that handles that, especially in the summertime, it’s just a matter of volume. We get so many samples, and trying to process everything takes some time.

When you’re doing this testing, and it comes back positive for a certain class of drugs, are you able to differentiate between specific drugs within a class?

Absolutely, yes. We would only report findings that we can present with legally and scientifically defensible data. That means that we have unequivocally identified the analyte as to exactly what it is. You would never see a report that says, “The sample contains a barbiturate.” It would say exactly which compound was detected. 

Is there any ability on your end to determine chronicity in a hair sample, or does it read as present or not present?

It can be basically how the client wants it. I’d have to refer you back to USEF because I’m not quite sure how they will address that. But I can tell you that chronicity can be affected by the length of hair that we’re testing. The longer the hair strand, the further back in time we’re going. 

We can’t limit it down to the hour—or really not even to the day—because growth rates of equine hair vary with seasonality, diet and just genetics. But you can limit the rough time period that was being tested.

I would not want to swear under oath that I can pinpoint it to a certain day, but I think that in a time period with a relevant margin of error, you could say, “Well, we tested this amount from the root, and based on average growth patterns, it’s acceptable to say that we’re within plus or minus three days or five days of this time period.” I do believe that is possible.

What kind of window are we talking about in testing for, say, barbiturates? What is the longest possible time frame you’re looking at?

It can go back as long as the hair is long. We have seen hair samples—not in the show world—where, instead of mane, they have submitted tail hair, which of course can get quite long. So, depending on how you section that hair, you might be able to look back 12 months, 18 months.

Do you prefer tail hair versus mane hair for that reason?

Generally speaking, our preference is for mane hair. The growth tends to be more stable within the mane rather than the tail. And the additional problems with tail hair can be—first of all, I imagine that the horse might be more sensitive to having tail hair yanked out, but there’s also the concern about fecal contamination. There are some natural products that can be metabolized into certain steroid-like compounds, and that’s just an interference that we don’t need during our testing.      

So mane hair is just generally cleaner. Of course, then you have the problem when people are roaching manes. I don’t know how often that happens [with] show horses, but sometimes the limitation is that people cut the mane too short. In that case, tail is definitely an option.

If this kind of testing is going to become typical at shows, do you think it’s something we’re going to see more commonly in pre-purchase exams?

I think what you will find with a lot of the accredited labs is there is a degree of reluctance on the laboratories’ part to accept samples from private parties, because some of our accreditation requirements basically say, “Thou shalt not test private party’s hair.” 

What laboratories have to be cautious about is that the test can be misused. You could do some experimentation on your horse and submit samples to see what the lab can and cannot detect. 

It’s an unfortunate thing, because I can completely sympathize with people wanting to be sure when they’re purchasing a new horse. Generally speaking, we only accept samples from official organizations and regulatory bodies, to avoid any sort of testing of laboratory capabilities.

It’s an unfortunate fact of life that a very small number of bad actors exist. 

Until now, USEF was looking for drugs that would typically be administered to a performance horse. Does that describe why they weren’t interested in using hair testing to this time to this date?

It’s good to mention that, at this point, most uses of the hair test are restricted to the detection of drugs that have no business being in a horse—in a performance horse, or any horse, for that matter. It’s not something that you would use to look for therapeutic medications or medications that have a legitimate application within the horse. 

Is there anything else I haven’t asked about hair testing that you think is important for readers to understand?

I understand that it’s always nerve-wracking when new tests are kind of unleashed on an industry, but I do want to emphasize, and I hope I have made that clear, that the testing is well established. This is not new technology. 

We are very conscious of the fact that the test results have a big impact on the people involved with that test, and we make every effort to ensure that the testing is scientifically impeccable—that it’s legally defensible. We take this work very seriously, and most of us are always willing and open to explaining as much as we possibly can about how the testing works. That’s the biggest takeaway: Take some small comfort in the fact that this is a very serious industry. We take our work very seriously. 

What is the cost of urine and blood testing compared to hair testing? 

It’s similar. It’s all in the same ballpark for pricing. It depends. With blood and urine, the scope of testing is much wider than it is in the hair sample, and then there are other factors, such as the required turnaround time, the positive rate. All of those factors come into play. 

Most of the labs that do this kind of testing, the labs will supply the collection supplies, or in some cases, pay for shipping of the samples back and forth. So there are other costs involved beyond the actual testing itself.

Can you describe, in layman’s terms, what it looks like to test a hair sample?

Envision what for humans would be a fairly considerable chunk of hair. We prefer for hair to be pulled, meaning we get follicles with it. We’re looking for the circumference of a No. 2 pencil; that’s about the thickness of the hair bundle that we ask for. 

The first thing that is done, of course, is verifying the integrity of the sample. So we look for certain conditions: The hair has to be dry. There can’t be any indication of chemical treatment of the hair. We have seen that in some samples where the hair comes smelling of chemicals, or you can tell it’s been exposed to chemicals, the hair is brittle and breaks off. So that hair sample would be rejected for testing. 

We, of course, expect that the sample is submitted in a sealed envelope, properly identified with a sample ID number. Also, we do not receive identifying information about the horse or associated humans with that horse. We just receive an anonymous sample number that’s entered in our records. We then then assign a laboratory identification number, and at that point, the sample is cleared for testing, the first step of which is actually a hair wash. 

We remove an amount of sample that is appropriate for the test that we’re running with a smaller number of strands. We actually measured it out one time: It’s about 250 individual hair strands that are used, that are washed to remove any sort of external contaminants. They are then dried, and the hair is inserted into a special tube that contains ceramic beads. The tube is sealed and placed into an instrument that submits the hair sample and the ceramic beads to high speeds. It agitates the beads, and the beads pulverize the hair. So what goes in are full-length hair strands, and what you end up with is a very fine hair powder that is then measured out on an analytical balance. 

We use about 50 milligrams per test of this powder, and that powder is then subjected to extraction procedures that are designed to eliminate what we would consider “junk.” So anything that we don’t want in our test, we’re trying to eliminate, and any potential drug that may be in the hair, we’re trying to concentrate; we achieve that through chemical extraction procedures. 

The hair, or the extract from this hair powder, undergoes several chemical reagent additions. It’s then dried down and re-diluted in a solvent placed inside a small vial that is loaded on an instrument called a liquid chromatograph tandem mass spectrometer. It’s kind of the gold standard for drug testing. 

That instrument then subjects the extract that is in the vial to a special program that is designed to target a certain number of drugs in that sample, and it provides us with a data printout. The run is not instantaneous. Usually we load a few hundred samples on this instrument at a time, but then each run takes a certain amount of time to go through the instrument and be analyzed. That run time varies depending on the drug classes that we’re looking for. It can be anywhere from five minutes to 15 minutes per sample. 

At the end of that, we have chemists that examine this data for any indication of the presence of a substance that is not supposed to be there. 

What this initial screening test is supposed to do is to rapidly identify to us which samples appear to be clean—so there’s no indication of anything being present—and which samples require a second look. 

Say that we receive 50 samples from a certain event or racetrack. At the end of the screening test, 45 of them might be released as clear, and five of them are going to say pending. That “pending” means that we’re doing additional testing to determine unequivocally, and in a legally defensible manner, that there is a prohibited substance present in that sample.

To do that, we essentially repeat that same process. We take a fresh sample from that original hair bundle, and we start all over, except this time—instead of on a number of different drugs—we’re focusing in on whatever the specific indication was in the screening test.

As complicated as it sounds, I like to say that “chemistry is like cooking; you just don’t lick a spoon.” I think that is really kind of true. It’s actually a very straightforward process, and it makes sense when you watch it being done.